CA2451528C - Novel aminopeptidase derived from bacillus licheniformis, gene encoding the aminopeptidase, expression vector containing the gene, transformant and method for preparation thereof - Google Patents

Abstract

The present invention relates to a novel aminopeptidase derived from Bacillus licheniformis, a gene encoding the aminopeptidase, an expression vector containing the gene, a cell transformant transfected with the expression vector and a process for preparing a natural type protein using the enzyme. More particularly, the present invention relates to a gene encoding an aminopeptidase which is cloned and manufactured using the recombinant DNA technique, an expression vector containing the gene, a cell transformant transfected with the expression vector and a recombinant aminopeptidase which is necessary to produce a recombinant human growth hormone in a natural type protein and can be expressed in a high yield more stably and advantageously, compared with conventional methods for the purification.

Description

NOVEL AMINOPEPTIDASE DERIVED FROM BACILLUS LICHENIFORMIS, GENE
ENCODING THE AMINOPEPTIDASE, EXPRESSION VECTOR CONTAINING THE
GENE, TRANSFORMANT AND METHOD FOR PREPARATION THEREOF

TECHNICAL FIELD

The present invention relates to novel aminopeptidase derived from Bacillus licheniformis, a gene encoding the aminopeptidase, an expression vector containing the gene, a transformant transfected with the expression vector and a process for preparing a natural type protein using thereof. More particularly, the present invention relates to a gene encoding an aminopeptidase which has an advantage that the protein can be more stably expressed in a high yield, by cloning the gene encoding the aminopeptidase which is necessary to produce a recombinant human growth hormone in natural type protein and producing the aminopeptidase by using the recombinant DNA
technology instead of conventional purification method, an expression vector containing the gene and a recombinant aminopeptidase.

BACKGROUND ART

Generally, recombinant proteins are produced as non-natural types when they are expressed in a large scale through the gene manipulation in microbes. In detail, most of the recombinant proteins have the initiating methionine(Met) due to incomplete treatment at amino terminus of the proteins. The proteins containing the N-terminal methionine may induce immunogenic reactions in human and animal bodies or may not express effectively their original function because of its unstable form. Therefore, it is very important to develop a process for preparing a natural type protein from proteins which produced by the recombinant DNA
technology(See Nature, 1987, 326, 315; J. Bacteriol., 1987, 169, 751-757; Bio/Technology, 1990, 8, 1036-1040; Appl. Microbiol.
Biotechnol., 1991, 36, 211-215).

A human growth hormone(hereinafter refer to as the "HGH") is a polypeptide composed of 191 amino acid residues, having 22,125 kDa of molecular weight and containing Phe-Pro-Thr sequence at amino terminus. And the HGH is produced in human pituitary gland and used mostly to improve dwarfism (See Raben, M. S., J. Clin.

Endocr., 1958, 18, 901). Up to now, the HGH has been extracted and purified from pituitary glands of the dead, but has not been provided for all the patients due to the limited supply. Recently, many researchers have attempted to produce a HGH in Escherichia coli and yeast by using the recombinant DNA technology and to utilize this HGH clinically(in case of Escherichia coli, See Korean Patent Publication No. 89-1244 and No. 87-701; Korean Patent Laid-open No. 87-2258 and No. 84-8695; in case of yeast, See Korean Patent Publication No. 92-99_ Korean Patent Laid-open No. 90-9973 and No. 90-9976).

However, the recombinant HGH produced by the above recombinant DNA technology has an extra methionine residue, corresponding to initiation codon, which is added at the N-terminus during protein synthesis although the natural type HGH is composed of 191 amino acids without methionine. Thus the recombinant HGH
which is composed of 192 amino acids and contains Met-Phe-Pro-Thr sequence at the N-terminus. In respect of biological activity, the methionyl HGH exhibit the same biological activity as that of the natural type of HGH(Moore, J. A., Endocrinology, 1988, 122, 2920-2926) and any side-effect caused from the extra methionine has not been reported. However, it is reported that antibodies against the HGH having the extra methionine may be generated due to the extra methionine residue, and its possibility of the antibodies generation is higher than that of the natural type protein (Lancet, 1986, March 29, 697).

Hence, there are several approaches to produce the natural type HGH without the N-terminal methionine. In particular, a method has been developed to obtain the natural type HGH, which comprises fusing the N-terminus of HGH to the C-terminus of another protein and cutting the fusion protein with a specific protease (See PCT
International Application WO 89/12678; European Patent Application EP 20209; European Patent EP 321940); and a method also has been attempted to obtain the natural type HGH from culture media, which comprises expressing a recombinant HGH in cell cytoplasm, secreting it out of cell and removing the methionine residue during secretion (See European Patent EP 008832; USA Patent USP 4755465;
Japanese Patent JP 01273591; European Patent Application EP 306673;

Korean Patent Application No. 92-10932). But, there are some disadvantages in these methods illustrated above. Precisely, it is required of complicated manipulation such as preparing expression vector and transforming host cells and fermentation condition should be optimized in each case.

On the other hand, a method has been exploited in case of recombinant proteins containing an extra amino acid at the N-terminus, which uses an aminopeptidase to remove the N-terminal amino acid and prepares natural type proteins by simple process. In particularly, the natural type of HGH can be prepared by using a specific aminopeptidase which selectively removes N-terminal methionine from the methionyl HGH purified through conventional methods in order to produce the natural type HGH (See PCT
International Application WO 86/04609 and WO 86/204527 Al).
Presently, several aminopeptidases which can be used to prepare a natural type HGH have been reported, such as an aminopeptidase purified from Aeromonas proteolytica (See PCT
Internatinal Application WO 86/01229; European Patent EP 0489711, A3, BTG company; Prescott and Wilks, Method in Enzymology, 1976, 44, 530-543), an aminopeptidase purified from pig kidney (See PCT
International Application WO 86/204527 Al; Bio/Technology, 1987, 5, 824-827, Takeda company), an dipeptidyl aminopeptidase purified from Dictyostelium discoidium (See European Patent EP 557076; USA
Patent USP 5126249, Al, Eli Lilly company), and an aminopeptidase from Streptomyces thermonitripican were reported.

In order to attain the above object, the aminopeptidase should react with only extra amino acid residues of recombinant proteins at the N-terminus, not with naturally occurring sequences of amino acids in order to prepare natural type proteins. Namely, when N-terminal amino acid sequences are X-Y-Z in a natural type protein and Met-X-Y-Z- containing an extra methionine in recombinant protein respectively, the aminopeptidase should remove only the methionine residue without reacting other amino acids such as (X-Y-Z-) to prepare the recombinant protein having the same amino acid sequence as that of the natural type protein. Therefore, the aminopeptidase satisfying the above conditions should be compatible with its substrate specificity depending upon the target protein for industrial applications. For industrial use, the enzyme also exhibits very high specific activity outstandingly.

Presently, more than several tens of aminopeptidases have been purified from microorganisms and the like and reported. In common, most of aminopeptidases need metal ions such as calcium, zinc or the like in order to be activated. These aminopeptidases have various activities according to molecular weight, essential metal ions, optimal conditions of the reaction, substrate specificity and also according to microorganisms, although they have common activities in view of the digestion of the amino acid residue at N-terminus of the protein (See FEMS Microbiol. Rev., 1996, 18, 319-344) . Such an aminopeptidase is classified to an exopeptidase removing amino acids from the N-terminus of substrate proteins gradually.

Precisely, these aminopeptidases have been reported and purified, especially from Bacillus genus, for example Bacillus subtilis (See Arch. Biochem. Biophys., 1979, 197, 63-77; Arch.

Biochem. Biophys., 202, 540-545, 1980; J. Biochem., 1994, 107, 603-607; Japanese Patent JP 03285684, Diacel-Chem company), Bacillus stearothermophilus (See Meth. Enzymol., 1970, 19, 544-552; Biochem.

Biophys. Acta, 1976, 438, 212-220; European Patent EP 101653, Unitika company), Bacillus thuringensis (Biokhimiya, 1984, 49, 1899-1907), Bacillus licheniformis (Arch. Biochem. Biophys., 1978, 186, 383-391; Mikrobiol. Zh., 1989, 51, 49-52) and so on.

In the references mentioned above, the aminopeptidases are purified and analyzed for their enzymatic properties by using leucine-p-nitroanilide, and several dipeptides or the like as a substrate. However, oligopeptides and proteins containing Met-X-Pro sequence at the N-terminus have not been used to examine the enzymatic activity. Therefore, it has not been expected that these aminopeptidases can be used for removal of the extra methionine from recombinant proteins containing Met-X-Pro sequence at the N-terminus.

Furthermore, the aminopeptidases derived from Bacillus licheniformis have been illustrated as follows. Rodriguez et al.
have purified the aminopeptidase derived from Bacillus licheniformis ATCC 12759 and examined its enzymatic property. Also, the inventors of the present invention have disclosed the method for preparing a natural type human growth hormone using the aminopeptidase derived from Bacillus licheniformis, the characterization of the purified aminopeptidase in its enzymatic properties and its amino acid sequence at N-terminus (Korean Patent Laid-open No. 1998-071239). In this invention, for producing a natural type protein in a large scale through recombinant DNA

technologies, the inventors of the present invention have reported that the aminopeptidase could remove only the extra methionine residue from synthetic substrates, oligopeptides, proteins and the like and recognize the specific amino acid sequence of Met-X-Pro (hereinafter, X denotes any amino acid regardless of its kind) and thus be suitable for the preparation of the natural type human growth hormone (Korean Patent Laid-open No. 1998-071239) . These conventional methods have elucidated that the aminopeptidase can be produced from Bacillus licheniformis and exploited to produce a natural type recombinant protein. Besides, they have provided only the information about the partial amino acid sequence of the N-terminus and the total gene of aminopeptidase has not determined yet.

Hence, the inventors of the present invention have tried to obtain a novel aminopeptidase for producing a natural type recombinant protein. Concretely, we have cloned a gene for an aminopeptidase derived from Bacillus licheniformis, determined its nucleotide sequence and amino acid sequence and expressed the cloned aminopeptidase gene in recombinant microorganisms. Then, the recombinant protein was identified to have aminopeptidase activities, to be useful in the other enzymatic reactions as well as the preparation of natural type proteins. Consequently, the present invention has been completed successfully.

DISCLOSURE OF INVENTION

The object of the present invention is to provide a novel aminopeptidase derived from Bacillus licheniformis, a gene encoding the aminopeptidase, an expression vector containing the gene, a cell transformant transfected with the expression vector and a process for preparing a natural type protein using the enzyme, which can be used to manufacture a recombinant protein as well as applied to other enzymatic reactions usefully.

In order to accomplish the object described above, the present invention provides an aminopeptidase derived from Bacillus licheniformis.

Precisely, the aminopeptidase has an amino acid sequence selected from the group consisting of the full length of SEQ. ID
NO: 1 and the amino acid sequences which are deletion of one or more amino acids from SEQ. ID NO: 1.

Preferably, the aminopeptidase has an amino acid sequence selected from the group consisting of the amino acid sequences which are deletion of one or more amino acids from SEQ ID NO:l at the amino terminus, carboxy terminus or both termini.

More preferably, the aminopeptidase has an amino acid sequence which is selected from the group consisting of: the amino acid sequence having the deletion of amino acids 1 through 30 from SEQ ID NO:l; the amino acid sequence having the deletion of amino acids 1 through 39 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 40 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 41 from SEQ ID NO:l; and the amino acid sequence having the deletion of amino acids 1 through 42 from SEQ ID NO:1.

Besides, the aminopeptidase has the amino acid sequence which is the deletion of amino acids 444 through 449 from SEQ. ID NO: 1 at the carboxylic terminus.

In addition, the present invention provides a gene encoding the aminopeptidase derived from Bacillus licheniformis.

Precisely, the gene encoding the aminopeptidase has a nucleotide sequence that encodes an aminopeptidase selected from the group consisting of SEQ ID NO:l, or a fragment thereof wherein said fragment is selected from the group consisting of: the amino acid sequence having the deletion of amino acids 1 through 30 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 39 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 40 from SEQ ID NO:l; the amino acid sequence having the deletion of amino acids 1 through 41 from SEQ ID NO:l; the amino acid sequence having the deletion of amino acids 1 through 30 and 444 through 449 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 39 and 444 through 449 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 40 and 444 through 449 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 41 and 444 through 449 from SEQ ID NO:1; and the amino acid sequence having the deletion of amino acids 444 through 449 from SEQ ID NO:1.

In addition, the present invention provides an expression vector pLAP132 which contains the gene encoding the aminopeptidase with the nucleotide sequence in the full length of SEQ. ID NO: 2.

Besides, the present invention provides an Escherichia coli transformant XLOLR/LAP132 which is transfected with the expression vector pLAP132 (accession number: KCTC 1000 BP).

Furthermore, the present invention provides a process for preparing a natural type protein which comprises the steps as follows: (1) purifying a recombinant proteins containing Met-X-Pro sequence at the amino terminus; (2) adding an aminopeptidase of the above-mentioned aminopeptidases to said purified recombinant protein; and (3) digesting Met-X-Pro sequence at the amino terminus of the recombinant protein by using the aminopeptidase.

At this moment, X of the Met-X-Pro can be any kind of amino acid.
BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

FIG. 1 depicts the determination of amino acid sequences in peptide fragments of the aminopeptidase obtained after treating trypsin.

FIG. 2 depicts the analysis of the aminopeptidase derived from Bacillus licheniformis and expressed from Escherichia coli transformant by performing SDS-polyacrylamide gel electrophoresis.

FIG. 3 depicts the analysis of the aminopeptidase derived from Bacillus licheniformis and expressed from Bacillus subtilis transformant by performing SDS-polyacrylamide gel electrophoresis.

FIG. 4 depicts the examination of enzymatic activities in the aminopeptidase derived from Bacillus licheniformis and expressed from Bacillus subtilis transformant schematically.

BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be illustrated more clearly as follows.

The aminopeptidase of the present invention is identified to have enzymatic activities regardless of whether or not signal peptide(the sequence consisting of the 1st amino acid through the 30th amino acid in SEQ. ID NO: 1) is deleted. Although some amino acids at the amino terminus and at the carboxy terminus are cut and deleted in addition to the deletion of the signal peptide, the enzymatic activities are maintained. Therefore, the aminopeptidase containing the amino acid sequence of SEQ. ID NO:
1 as well as the aminopeptidases with a partial deletion at the amino terminus or at the carboxy terminus from the amino acid sequence of SEQ. ID NO: 1 can be within the scope of the present invention.

Preferably, the aminopepdidase of the present invention is selected from a group of aminopeptidases having a deletion at the amino terminus of SEQ ID NO:1 wherein said amino acid sequence is selected from the group consisting of: the amino acid sequence having the deletion of amino acids 1 through 30 from SEQ ID NO:1;
the amino acid sequence having the deletion of amino acids 1 through 39 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 40 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 41 from SEQ ID NO:1; the amino acid sequence having the deletion of amino acids 1 through 42 from SEQ ID NO:1. More preferably, the aminopeptidase of the present invention has the deletion of amino acids 1 through 30 from SEQ ID NO:1.

Preferably, the aminopeptidase of the present invention has the amino acid sequence of SEQ ID NO:1 having a deletion of amino acids 444 through 449 at the carboxy terminus.

More preferably, the aminopeptidase has the amino acid sequence of SEQ. ID NO: 1 having a deletion of amino acids 1 through 42 and 444 through 449.

The genes of the present invention encoding the aminopeptidase derived from Bacillus licheniformis can include the gene encoding amino acid sequence of SEQ. ID NO: 1, or the gene encoding all of the deleted form of aminopeptidase mentioned above and the gene of SEQ. ID NO: 2.

In order to investigate functions and enzymatic activities of the aminopeptidase, the following procedure is accomplished by using the protein containing the amino acid sequence of SEQ. ID NO:

1, its genes and its polypeptide in a deleted form respectively.

In order to elucidate polypeptides having enzymatic activities of the aminopeptidase derived from Bacillus licheniformis, a gene encoding the aminopeptidase is cloned from the chromosomal DNA of Bacillus licheniformis. Concretely, for cloning genes, polypeptides having the enzymatic activities of the aminopeptidase derived from Bacillus licheniformis are purified, digested with trypsin so as to collect a number of peptide fragments and then determined the amino acid sequences. The information of the amino acid sequences is exploited to synthesize oligonucleotides for use of primers and then DNA fragments corresponding to a part of the aminopeptidase gene are amplified.
Afterward, by utilizing these DNA fragments for probes, the aminopeptidase gene can be found from the chromosomal library derived from Bacillus licheniformis.

The genes of aminopeptidase observed above are examined to determine the nucleotide sequences with the DNA sequence analyzer as shown in SEQ. ID NO: 2, and the deduced DNA sequence from the genetic information of SEQ. ID NO: 1 is identified to have exactly the same amino acid sequence of the aminopeptidase purified from natural host cells. As a result, the aminopeptidase polypeptide obtained according to the present invention is screened by using data base for genetic informations and this gene is identified a novel DNA sequence that have never reported and have a enzymatic activities of the aminopeptidase when it is expressed in recombinant microbes.

Then, the matured aminopeptidase is also detected to be digested partially. In order to investigate the composition of the aminopeptidase at the carboxy terminus, the aminopeptidase purified from the recombinant bacterial transformant and the aminopeptidase purified from natural host cells, Bacillus licheniformis, are examined to determine molecular weights with mass spectrometry.
Consequently, in both 2 cases 6 amino acid residues are verified to be cut from the carboxy terminus.

The aminopeptidase according to the present invention is confirmed to become mature that the aminopeptidase were expressed in the cell and then, the signal peptide was deleted at the amino terminus during the extracellular secretion. Besides, the matured aminopeptidase is also digested partially.

Hence, the aminopeptidase polypeptide according to the present invention maintains enzymatic activities with presence of the signal peptide and even partially being cut at the amino terminus or at the carboxy terminus with absence of the signal peptide.

EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

<Example 1> Cloning of aminopeptidase gene derived from Bacillus licheniformis (1-1) Partial determination of amino acid sequence in aminopeptidase purified from Bacillus licheniformis The present inventors have performed the procedure as follows in order to purify aminopeptidase protein from Bacillus licheniformis.

Sterilized media containing tryptone 10 g, yeast extract 5 g, NaCl 10 g per 1 L were prepared in flasks and Bacillus licheniformis KCTC 3058 strain was inoculated so as to be cultivated for seed-culture at 40 C, 120 rpm for about 16 hours.
The seed culture broth obtained above was inoculated again to new media and cultivated at 40 C, 200 - 400 rpm agitation, more than 30 of dissolved oxygen. Then, the concentrations of glucose were measured with an hour interval and the cultivation was completed when the activity of aminopeptidase reached more than 35 U/mk . In order to cultivate cells, culture broth containing peptone 2 kg, yeast extract 6 kg, potassium phosphate 4 kg, NaCl 1 kg, SAG, MgSO4-7H20 15 g, FeSO477H20 1.53 g, ZnSO4*7H20 1.53 g, MnS04 1.53 g, and glucose 10 kg per distilled water 200 L in 400 L fermentor was prepared in a sterilized state. After the cultivation was completed, continuous centrifuge was utilized to remove cell debris and recovered the supernatants. The supernatant obtained above was concentrated with a concentrator and then ZnS04 was added to reach about 0.3 mM. Through 3 steps of column chromatography, aminopeptidase was purified from the concentrated solution. As a chromatography, SP-SepharoseTM FF, SephacrylTM S-200, and DEAE-SepharoseTM FF were utilized in turns. The aminopeptidase purified by using the above procedure was identified to have more than 95%
of purity when analyzed with the reverse phase high pressure chromatography.

The purified aminopeptidase was precipitated by using 10%
trichloroacetic acid (TCA), washed with acetone and dried. The dried protein precipitate was dissolved with 8 M urea and 0.4 M
ammonium bicarbonate and treated with dithiothreitol(DTT) and iodoacetamide in turns so that disulfide bonds in the aminopeptidase were cleaved. The treated sample was dissolved again in distilled water, added about 0.05mg of trypsin per 2 mg of aminopeptidase and treated at 37 C for about 8 hours. The sample treated with trypsin was injected into the reverse phase high pressure chromatography (RP-HPLC) and collected to fractions of major peptide peaks. In the reverse phase chromatography, VydacTM
C18 reverse phase column was adopted and a linear concentration gradient using solvent A (highly purified water containing 0.05%
trifluoroacetic acid(TFA)) and solvent B (highly purified water containing 0.05% TFA and 80% acetonitrile) was applied. At this moment, the analysis was performed in 0.5 m~ /min of flow velocity and chromatogram was obtained by UV absorbance at 214 nm. Through this procedure, more than 10 peaks were obtained. The resulted peptide sample was analyzed with a mass spectrometry respectively and peptides composed of more than 15 amino acids were selected.
The selected sample was determined the amino acid sequence with the amino acid sequence analyzer. FIG. 1 depicted the amino acid sequence of the selected peptide by using 1 character denoting an amino acid. Each peptide was numbered in orders arbitrarily in accordance with elution time through reverse phase chromatography and the arrow part depicted in FIG. 1 was used to give information for synthesizing the primers of oligonucleotides. The sample denoted as T4 among these peptide samples were deduced to contain 2 different peptides coincidently since 2 amino acids appeared at the same time in each cycle by performing the sequence analysis of amino acids. Besides, among these peptide samples, the same sequence of amino acids is contained in between T6 and T9, T11 and T13 and T16 and T17. Precisely, T4 described in SEQ. ID NO: 4 and 5, T6 in SEQ. ID NO: 6, T7 in SEQ. ID NO: 7, T9 in SEQ. ID NO: 8, Tll in SEQ. ID NO: 9, T13 in SEQ. ID NO: 10, T16 in SEQ. ID NO:
11, T17 in SEQ. ID NO: 12 respectively, which is disclosed in Sequence List independently.

(1-2) Cloning of aminopeptidase gene and determination of nucleotide sequence By using the peptide information elucidated in Example 1(1-1), oligonucleotide primers for PCR of aminopeptidase genes were synthesized. Precisely, 5'-upstream primer(LAP-5) described in SEQ.
ID NO: 13 and 3'-downstream primer(LAP-3) described in of SEQ. ID
NO: 14 were utilized, which were manufactured by using the amino acid sequences described in SEQ. ID NO: 15 and SEQ. ID NO: 16 among amino acid sequences determined in Example 1 (1-1) . PCR was performed 32 times repeatedly with the DNA thermal cycler;
denaturation at 94 C for 30 seconds, annealing at 40 C for 45 seconds, and extension at 72 C for 1 minute and chromosomal DNA of Bacillus licheniformis was used as a template. As a result, DNA
fragment with about 390 bp size was obtained.

In the meantime, genomic DNA of Bacillus licheniformis was made by exploiting Murray and Thompson's method and digested partially with the restriction enzyme Sau3A and separated it through 0.8% agarose gel then isolated the DNA fragments corresponding to 2 - 3kb. The DNA fragment was ligated into A ZAP

expression vector (Stratagene, LaJolla, USA) digested with BamHI
and added to packaging extract. Then, the packaging mixture was transferred to E. coli XL-l Blue MRF'. This library filter prepared above was screened by using 32P-labelled PCR fragment and detected for clones containing aminopeptidase gene. As a result, the selected clone was verified to include DNA insert with about 2.6 kb size and named it "LAP 132" clone.

The nucleotide sequence of LAP 132 was analyzed and the result was described in SEQ. ID NO: 2. In detail, it was composed of ATG initiation codon starting at 192 nucleotide and TAA

termination codon at 1,539 and contained 1,347 bp ORF (open reading frame) as shown in SEQ. ID NO: 2 and in SEQ. ID NO: 3. The nucleotide was identified to encode 449 amino acids. The amino acid sequence encoded from the nucleotide sequence information elucidated above, were identical completely with the result determined by the amino acid sequence analysis of peptide fragments derived from aminopeptidase purified above. The domain of amino terminus of the aminopeptidase according to the present invention contained hydrophobic amino acid residues contiguous to cationic amino acid residues, which was similar to the signal sequence needed for the secretion. Thus, the aminopeptidase was deduced to be digested in between 30th alanine and 31st alanine when secreted.
But, in Korean Patent Laid-open No. 1998-071239, the aminopeptidase was disclosed that amino terminus mainly starts from amino acid sequences described in SEQ. ID NO: 17 and heterogeneous type forms of aminopeptidases of which their cleaved sites are somewhat different with the present invention. This difference was deduced to be provoked by digesting with the other extracellular protease after the aminopeptidase was secreted from original strains. The aminopeptidase according to the present invention was compared in the amino acid sequence with other aminopeptidase recorded in Genebank by using BLAST searching. As a result, it was shown to have 62% homology with the aminopeptidase derived from Bacillus subtilis, and 58% homology with aminopeptidase derived from Bacillus halodurans. The aminopeptidase according to the present invention identified as a new aminopeptidase not reported previously.

The inventors of the present invention have transformed the aminopeptidase gene, "LAP 132" derived from Bacillus licheniformis, into E. coli XLOLR strain and have named with "E. coli XLOLR/LAP
132". They have deposited with International Deposit Organization, the Korean Collection for Type Cultures (KCTC) of the Korean Research Institute of Bioscience and Biotechnology (KRIBB), Republic of Korea on April 26, 2001, and identified as accession number, KCTC 1000 BP.

<Example 2>, Gene expression of aminopeptidase in Escherichia coli transformant The aminopeptidase gene cloned according to the present invention was subcloned into the expression vector pBK-CMV
(Stratagene, USA) and named the expression vector "pLAP32" so as to be used as a source of aminopeptidase gene. Then, the PCR fragment with about 1.2 kb size containing a region encoding aminopeptidase was subcloned into the vector pET11a (Stratagene, USA) and named the expression vector pETLAP45. The expression vector was transformed into E. coli BL21(DE3) and exploited it to express a fused protein attaching His-tag peptide. The expression of aminopeptidase in the expression system described above was verified by performing SDS-PAGE. As illustrated in FIG. 2, the molecular weight of aminopeptidase was examined to reach about 45 kDa onto SDS-PAGE and identical to that deduced from its gene. In FIG. 2, lane M depicted a standard marker of molecular weights;
lane 1, E. coli transformed into the expression vector pET11a (not inducing the expression); lane 2, E. coli transformed into the expression vector pET11a (inducing the activation of T7 promoter);
lane 3, E. coli transformed into the expression vector pETLAP45 (not included the expression); and lane 4, E. coli transformed into the expression vector pETLAP45 (inducing the activation of T7 promoter). The arrow depicted in FIG.2 denoted the aminopeptidase.
The inventors of the present invention have tried to detect enzymatic activities of aminopeptidase secreted from E. coli transformant after expression the gene. Above all, when the E. coli transformant was sonicated and analyzed, the enzymatic activities of the aminopeptidase from the E.coli transformant were found in soluble fractions. As a result, the aminopeptidase according to the present invention was identified to have the same size with that deduced from the gene and to show the enzymatic activities.
<Example 3> Gene expression of aminopeptidase in Bacillus Subtilis transformant, sequence analysis of the purified aminopeptidase at the amino terminus and determination of its molecular weight (3-1) Gene expression of aminopeptidase in Bacillus licheniformis transformant In the present invention, DNA fragment digested with Hindlll-SacI, containing a region encoding aminopeptidase as well as promoter, 3'-untranslated region, having about 2.6 kb size and was subcloned into the Bacillus vector pRB373 and the resulted expression vector was named pRB373-LAP. The expression vector was transformed into Bacillus subtilis, and cultivated and then the cultured broth was analyzed by using SDS-PAGE. The aminopeptidase was observed to be secreted from the cultured solution and to have about 45 kDa of molecular weight, which was compatible with that from Bacillus licheniformis (See FIG. 3) . In FIG. 3, lane M

depicted a standard marker of molecular weight; lane 1, Bacillus subtilis transformed into the expression vector pRB373; lane 2, Bacillus subtilis transformed into the expression vector pRB373-LAP; and lane 3, aminopeptidase purified from Bacillus licheniformis. The arrow depicted in FIG. 3 denoted the aminopeptidase.

(3-2) Sequence analysis of the aminopeptidase polypeptide at the amino terminus expressed from Bacillus subtilis transformant In order to purify the aminopeptidase, recombinant Bacillus subtilis transformant containing the gene according to the present invention was cultivated and the cultured broth was separated by performing SP-SepharoseTM chromatography. The amino acid sequence at the amino terminus of the purified aminopeptidase was determined by the amino acid analyzer. As a result, it was verified that the sequence of aminopeptidase at the amino terminus was heterogeneous as found in the aminopeptidase derived from natural host cells and initiated with SEQ. ID NO: 17 mostly among those with SEQ. ID NO: 18. And the aminopeptidase initiated with Asn, Val and Glu also existed.

(3-3) Determination of molecular weight in aminopeptidase polypeptide expressed from recombinant Bacillus subtilis transformant The recombinant Bacillus subtilis transformant containing the aminopeptidase gene was cultivated and the cultured broth was exploited to purify the aminopeptidase by using SP-SepharoseTM
chromatography. The molecular weight of the aminopeptidase was examined with mass spectrometry and as a result, substances corresponding to 42,965 Da of molecular weight, 43,241 Da and 43,468 Da appeared. This result was compared with that obtained from the sequence analysis of amino acids in Example 3(3-2) and additional 6 amino acids was deduced to be removed at the carboxy terminus. That is to say, the polypeptide described in SEQ. ID NO:
1, the amino terminus started from SEQ. ID NO: 17 and the carboxy terminus ended to SEQ. ID NO: 19 had a theoretical molecular weight corresponding to about 43,241 Da. Then, another polypeptide added in 2 amino acids from the above had a molecular weight corresponding to 43,468 Da and the other polypeptide deleted in 2 amino acids had a molecular weight corresponding to 42,965 Da, which was identical to the results obtained from mass spectrometry exactly.

In the meantime, the aminopeptidase purified from a natural host cell, Bacillus licheniformis was examined by using mass spectrometry through the same procedure of Example 1 (1-1) and the same result with that of recombinant transformant was obtained.

<Example 4> Activity measurement of aminopeptidase and its deleted forms expressed in recombinant E. coli transformant and Bacillus subtilis transformant The inventors of the present invention measured the aminopeptidase activity using cell lysate solution sonicated in case of recombinant E. coli transformant and using cultured solution obtained in Example 3 (3-1) in case of recombinant Bacillus subtilis transformant.

Concretely, Pflleiderer's method was utilized in order to estimate enzymatic activities of the aminopeptidase produced in Example 2 and Example 3 (Pflleiderer, Meth. Enzymol., 1970, 19, 514-521) . 50 l of cultured solution was added into the mixture of 1 M Tris (pH 8.5)(950 l), and 0.1 M leucine-p-nitroanilide(20 l) in DMSO, reacted for 3 minutes at 60 C, added 100 l of 70% acetic acid, ended the reaction and detected the absorbance at 405 nm.

Consequently, as shown in FIG 4, there was a precise difference in between Bacillus subtilis strain transformed with the aminopeptidase gene and Bacillus subtilis strain containing the expression vector. In addition, the aminopeptidase purified from recombinant Bacillus subtilis was composed conincidently of deleted forms at the amino terminus or at the carboxy terminus as demonstrated in Example 3 (3-2) and (3-3) and the samples were also identified to have enzymatic activities. In FIG. 4, ^ depicted pRB373-LAP, Bacillus subtilis transformed with the expression vector containing the aminopeptidase gene; A depicted LG, Bacillus subtilis cultivated in LB culture broth; and = depicted pRB373, Bacillus subtilis transformed with the expression vector not containing an aminopeptidase gene.

INDUSTRIAL APPLICABILITY

As demonstrated clearly and confirmed above, according to the present invention the gene encoding the aminopeptidase derived from Bacillus licheniformis is cloned and expressed by using the recombinant bacterial transformant and is identified to be a novel gene not reported previously. The aminopeptidase purified and elucidated according to the present invention can be exploited usefully to manufacture natural type recombinant proteins as well as applied to other enzymatic reactions widely.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

SEQUENCE LISTING

<110> LG Life Sciences Ltd.
<120> Novel Aminopeptidase Derived from Bacilus Licheniformis, Gene Encoding the Aminopeptidase, Expression vector containing the Gene, Transformant and Method for Preparation Thereof <130> 863-107 <140> 2,451,528 <141> 2002-07-06 <150> KR 2002-0030798 <151> 2002-05-31 <150> KR 2001-0040268 <151> 2001-07-06 <160> 19 <170> Kopatentln 1.71 <210> 1 <211> 449 <212> PRT
<213> Bacillus licheniformis <220>
<221> SIGNAL
<222> (1)..(30) <220>
<221> DOMAIN
<222> (133)..(211) <223> PA (Protease associated) domain <220>
<221> DOMAIN
<222> (261)..(308) <223> Peptidase family M20/M25/M40 <400> 1 Met Lys Arg Lys Met met met Ile Gly Leu Ala Leu Ser Val Ile Ala Gly Gly Val Phe Ala Ala Gly Thr Gly Asn Ala Val Gln Ala Ala Pro Gln Glu Thr Ala Ile Ala Lys Asn Val Glu Lys Phe Ser Lys Lys Phe Asn Glu Asn Arg Ala Tyr Gln Thr Ile Tyr His Leu Ser Glu Thr Val Gly Pro Arg Val Thr Gly Thr Ala Glu Glu Lys Lys Ser Ala Ala Phe Ile Ala Ser Gln Met Lys Lys Ser Asn Leu Lys Val Thr Thr Gln Thr Phe Ser Ile Pro Asp Arg Leu Glu Gly Thr Leu Thr Val Gin Gly Asn Asn Val Pro Ser Arg Pro Ala Ala Gly Ser Ala Pro Thr Ala Ala Glu Gly Leu Ala Ala Pro Leu Tyr Asp Ala Gly Leu Gly Leu Pro Gly Asp Phe Thr Glu Glu Ala Arg Gly Lys Ile Ala Val Ile Leu Arg Gly Glu Leu Thr Phe Tyr Glu Lys Ala Lys Asn Ala Ala Asp Ala Gly Ala Ser Gly Val Ile Ile Tyr Asn Asn Val Asp Gly Leu Val Pro Leu Thr Pro Asn Leu Ser Gly Asn Lys val Asp Val Pro Val val Gly Val Lys Lys Glu Asp Gly Glu Lys Leu Leu Ser Glu Gln Glu Ala Ile Leu Lys Leu Lys Ala His Lys Asn Gln Thr Ser Gln Asn Val Ile Gly Val Arg Lys Ala Lys Gly Val Lys Asn Pro Asp Ile Val Tyr val Thr Ser His Tyr Asp Ser Val Pro Tyr Ala Pro Gly Ala Asn Asp Asn Ala Ser Gly Thr Ser Val Val Leu Glu Leu Ala Arg Ile Met Lys Thr Val Pro Ala Asp Lys Glu Ile Arg Phe Ile Thr Phe Gly Ala Glu Glu Ile Gly Leu Leu Gly Ser Arg His Tyr Val Ser Thr Leu Ser Glu Gln Glu Val Lys Arg Ser Val Ala Asn Phe Asn Leu Asp Met Val Ala Thr Ser Trp Glu Asn Ala Ser Gln Leu Tyr Ile Asn Thr Pro Asp Gly Ser Ala Asn Leu Val Trp Gln Leu Ser Lys Ala Ala ser Leu ser Leu Gly Lys Asp Val Leu Phe Leu His Gln Gly Gly Ser Ser Asp His Val Pro Phe His Glu Ala Gly Ile Asp Ser Ala Asn Phe Ile Trp Arg Glu Pro Gly Thr Gly Ala Leu Glu Pro Trp Tyr His Thr Pro Tyr Asp Thr Ile Glu His Ile Ser Lys Asp Arg Leu Lys Thr Ala Gly Gln Ile Ala Gly Thr Ala Val Tyr Asn Leu Thr Lys Lys Glu Asn Arg Thr Pro Ser Tyr Ser Ser Val Ala Gln 3b mise en contact d'un echantillon biologique preleve chez un patient, avec des anticorps diriges contre I'un ou plusieurs des peptides selon la revendication 9 et la detection des complexes immunologiques formes entre les antigenes de VIH-1 dans I'echantillon biologique et lesdits anticorps.
La presente invention a egalement pour objet un reactif de diagnostic d'un VIH-1 non-M non-O, caracterise en ce qu'il comprend une molecule d'acide nucleique telle que definie precedemment ou un peptide tel que defini precedemment.
La presente invention a egalement pour objet un procede de criblage et de typage d'un VIH-1 non-M non-O, caracterise en ce qu'il comprend la mise en contact de I'un quelconque des fragments nucleotidiques tels que definis precedemment avec I'acide nucleique du virus a typer et la detection de I'hybride forme.
La presente invention a egalement pour objet une trousse de diagnostic de VIH-1 non-M non-O, caracterisee en ce qu'elle inclut au moins un reactif tel que defini precedemment.
La presente invention a egalement pour objet la sequence nucleoti-dique complete de la souche telle que definie ci-dessus (SEQ ID N 1) ainsi que des fragments d'acide nucleique d'au moins 10 nucleotides, issus de ladite souche.
Parini ces fragments, on peut citer :
- LTR YBF 30 (SEQ ID N 2), - GAG YBF 30 (SEQ ID N 3) (gene gag), - POL YBF 30 (SEQ ID N 5) (gene poly, - VIF YBF 30 (SEQ ID N 7) (gene vrf), - VPR YBF 30 (SEQ ID N 9) (gene vpr), - VPU YBF 30 (SEQ ID N 11) (gene vpu), - TAT YBF 30 (SEQ ID N 1:3) (gene tat), - REV YBF 30 (SEQ ID N 15) (gene rev), <210> 2 <211> 2052 <212> DNA
<213> Bacillus licheniformis <220>
<221> RBS
<222> (179)..(184) <223> RBS candidate 1 <220>
<221> RBS
<222> (195)..(200) <223> RBS candidate 2 <220>
<221> CDS
<222> (192)..(1538) <223> CDS candidate 1 <220>
<221> mat-peptide <222> (282)..(1538) <220>
<221> Sig peptide <222> (192)..(281) <400> 2 gatcctgata attcggacgt attctaaaca gaaaaaaggc tcacgtcaag catcctatta 60 aacaaaaaaa cttttttatc aaacttcaaa ttactggtct atcgaatcat ttatcagatg 120 catgcaggat tcacccgctc agctgcgaat atctcttctc aggaaaacaa gaccatcaag 180 gaggtttatg t atg aag aga aaa atg atg atg atc gga ttg gcg 224 Met Lys Arg Lys Met Met Met Ile Gly Leu Ala cta tcc gta ata gca ggc ggc gtg ttc gcc get gga acg ggg aat get 272 Leu Ser Val Ile Ala Gly Gly Val Phe Ala Ala Gly Thr Gly Asn Ala gtt caa gcg gcg cct cag gaa aca gcc atc gca aaa aat gtc gaa aaa 320 Val On Ala Ala Pro Gln Glu Thr Ala Ile Ala Lys Asn Val Glu Lys ttc agc aaa aaa ttc aat gaa aac cgc gcc tat caa acg att tac cat 368 Phe Ser Lys Lys Phe Asn Glu Asn Arg Ala Tyr Gln Thr Ile Tyr His tta agc gaa acg gtc gga ccg cgt gtg aca ggc acg gcg gaa gaa aaa 416 Leu Ser Glu Thr Val Gly Pro Arg Val Thr Gly Thr Ala Glu Glu Lys aag agc gcc get ttc atc gcc tca cag atg aaa aaa tca aat ctg aaa 464 Lys Ser Ala Ala Phe Ile Ala Ser Gln Met Lys Lys Ser Asn Leu Lys gtg acc aca caa acc ttc agc ata cct gac cgg ctg gaa gga acg ctt 512 Val Thr Thr Gln Thr Phe Ser Ile Pro Asp Arg Leu Glu Gly Thr Leu acc gtt cag gga aat aac gtg cct tcg cgg cct gcc gcc ggt tcc gcc 560 Thr Val Gin Gly Asn Asn Val Pro Ser Arg Pro Ala Ala Gly Ser Ala ccg aca gca gca gaa ggc ctg gcc get cct ctc tat gat gcc ggc ctc 608 Pro Thr Ala Ala Glu Gly Leu Ala Ala Pro Leu Tyr Asp Ala Gly Leu ggc ctg cct ggc gac ttc acc gag gaa gcg aga ggc aaa atc gcc gtc 656 Gly Leu Pro Gly Asp Phe Thr Glu Glu Ala Arg Gly Lys Ile Ala Val att tta aga ggc gag ctg aca ttc tat gaa aaa gcg aaa aac get get 704 Ile Leu Arg Gly Glu Leu Thr Phe Tyr Glu Lys Ala Lys Asn Ala Ala gac gca g c gca agc g a gtg atc att tat aat aac gtc gac ggt ctc 752 Asp Ala Gly Ala Ser Gly Val Ile Ile Tyr Asn Asn Val Asp Gly Leu gtc cct ctg act ccg aat ctc agc gggt aat aaa gtc gat gtt ccg gta 800 Val Pro Leu Thr Pro Asn Leu Ser Gly Asn Lys Val Asp Val Pro Val gtc ggc gtc aaa aag gaa gac gga gaa aag ctg ctt tct gaa caa gaa 848 Val Gly Val Lys Lys Glu Asp Gly Glu Lys Leu Leu Ser Glu Gln Glu gcg atc ttg aag ctg aag get cat aaa aat caa aca tcg caa aac gta 896 Ala Ile Leu Lys Leu Lys Ala His Lys Asn Gln Thr Ser Gln Asn Val atc ggc gtc cgc aaa gca aaa ggt gtc aaa aat ccg gac atc gtg tat 944 Ile Gly Val Arg Lys Ala Lys Gly Val Lys Asn Pro Asp Ile Val Tyr gtg act tcg cat tat gac agc gtc cct tac get ccc gga gcc aat gac 992 Val Thr Ser His Tyr Asp Ser Val Pro Tyr Ala Pro Gly Ala Asn Asp aat gcc tcc ggc act tca gtc gtt ctt gaa ctg gcc cgg atc atg aag 1040 Asn Ala Ser Gly Thr Ser Val Val Leu Glu Leu Ala Arg Ile Met Lys acg gtt ccg gcc gac aaa gaa att cgc ttt att aca ttc g c gcc gaa 1088 Thr Val Pro Ala Asp Lys Glu Ile Arg Phe Ile Thr Phe Gly Ala Glu gaa atc ggt ctc ctc g 9a tcg cgc cat tat gtc agc acc ttg tca gag 1136 Glu Ile Gly Leu Leu Gly Ser Arg His Tyr Val Ser Thr Leu Ser Glu cag gaa gtc aaa cgg agc gtt gcc aac ttt aac tta gat atg gtg gcg 1184 Gln Glu Val Lys Arg Ser val Ala Asn Phe Asn Leu Asp Met val Ala aca agc tgg gaa aat get tca cag ctg tac atc aat aca cct gac g t 1232 Thr Ser Trp Glu Asn Ala Ser Gin Leu Tyr Ile Asn Thr Pro Asp BY

tca gca aac ctc gtc tgg cag cta agt aaa gcc get tct tta agc ctt 1280 Ser Ala Asn Leu Val Trp Gln Leu Ser Lys Ala Ala Ser Leu Ser Leu ggg aaa gac gta tta ttt tta cat caa ggc gga tca tcc gac cat gtc 1328 Gly Lys Asp Val Leu Phe Leu His Gin Gly Gly Ser Ser Asp His Val cca ttc cat gaa gcc ggc atc gac tca gcc aac ttc att tgg aga gag 1376 Pro Phe His Glu Ala Gly Ile Asp Ser Ala Asn Phe Ile Trp Arg Glu ccg gga aca ggt gca ttg gag cct tgg tac cac acc cct tac gac acg 1424 Pro Gly Thr Gly Ala Leu Glu Pro Trp Tyr His Thr Pro Tyr Asp Thr att gaa cac atc agc aaa gac agg ctg aaa aca gcc g a caa atc gcg 1472 Ile Glu His Ile Ser Lys Asp Arg Leu Lys Thr Ala Gly Gln Ile Ala g a aca gcc gtg tat aac ctg acc aag aaa gaa aac aga aca ccg tct 1520 Giy Thr Ala Val Tyr Asn Leu Thr Lys Lys Glu Asn Arg Thr Pro Ser tac agc tca gtc gcc caa to atattaaaaa ggagcagatc gattcaatct 1570 Tyr Ser Ser Val Ala Gln gctccttttt tataccgctt cttttcaatc cttcatcagc ttaataaacc tgaagctcat 1630 caaaacgctg ccgatggcaa ccacaatcat gaccaccccc agatcctgaa aatgacccgc 1690 ggttatttct tctccaaaca acagacccag aatgacggac gaaaagatcg agccaagata 1750 gcggcatgtt tggaagagcc ccgaagtggt tccgacgatg tccggcgggc ttgccgtaaa 1810 catggcggcc tggagggcga cattgccgag tccatagctg acccccagca aagagaggat 1870 gatgcctttc cacaacatcg gtgcatcgac aaaaaacaat gtgagcagga tagcgccggc 1930 tgccattaag caagaaccaa ttaaaacagg ctgcgtttca cctgaacggt caatccatgt 1990 cccgacgaaa ggcgaaatca atacgctcgt cccggacatg aacagcatca aaagacccgt 2050 cg 2052 <210> 3 <211> 449 <212> PRT
<213> Bacillus licheniformis <400> 3 Met Lys Arg Lys Met met met Ile Gly Leu Ala Leu Ser Val Ile Ala Gly Gly Val Phe Ala Ala Gly Thr Gly Asn Ala val Gln Ala Ala Pro Gln Glu Thr Ala Ile Ala Lys Asn Val Glu Lys Phe Ser Lys Lys Phe Asn Glu Asn Arg Ala Tyr Gln Thr Ile Tyr His Leu Ser Glu Thr Val Gly Pro Arg Val Thr Gly Thr Ala Glu Glu Lys Lys Ser Ala Ala Phe Ile Ala Ser Gln Met Lys Lys Ser Asn Leu Lys Val Thr Thr Gln Thr Phe Ser Ile Pro Asp Arg Leu Glu Gly Thr Leu Thr Val Gln Gly Asn Asn Val Pro ser Arg Pro Ala Ala Gly Ser Ala Pro Thr Ala Ala Glu Gly Leu Ala Ala Pro Leu Tyr Asp Ala Gly Leu Gly Leu Pro Gly Asp Phe Thr Glu Glu Ala Arg Gly Lys Ile Ala Val Ile Leu Arg Gly Glu Leu Thr Phe Tyr Glu Lys Ala Lys Asn Ala Ala Asp Ala Gly Ala Ser Gly Val Ile Ile Tyr Asn Asn Val Asp Gly Leu Val Pro Leu Thr Pro Asn Leu Ser Gly Asn Lys Val Asp Val Pro Val Val Gly val Lys LyS

Glu Asp Gly Glu Lys Leu Leu Ser Glu Gln Glu Ala Ile Leu Lys Leu Lys Ala His Lys Asn Gln Thr Ser Gln Asn Val Ile Gly val Arg Lys Ala Lys Gly val Lys Asn Pro ASp Ile val Tyr Val Thr Ser His Tyr Asp Ser Val Pro Tyr Ala Pro Gly Ala Asn Asp Asn Ala Ser Gly Thr Ser Val Val Leu Glu Leu Ala Arg Ile Met Lys Thr Val Pro Ala Asp Lys Glu Ile Arg Phe Ile Thr Phe Gly Ala Glu Glu Ile Gly Leu Leu Gly Ser Arg His Tyr val Ser Thr Leu Ser Glu Gln Glu Val Lys Arg Ser Val Ala Asn Phe Asn Leu ASP Met val Ala Thr Ser Trp Glu Asn Ala Ser Gln Leu Tyr Ile Asn Thr Pro Asp Gly Ser Ala Asn Leu Val Trp Gln Leu Ser Lys Ala Ala Ser Leu Ser Leu Gly Lys Asp Val Leu Phe Leu His Gin Gly Gly Ser Ser Asp His Val Pro Phe His Glu Ala Gly Ile Asp Ser Ala Asn Phe Ile Trp Arg Glu Pro Gly Thr Gly Ala Leu Glu Pro Trp Tyr His Thr Pro Tyr Asp Thr Ile Glu His Ile Ser Lys Asp Arg Leu Lys Thr Ala Gly Gln Ile Ala Gly Thr Ala Val Tyr Asn Leu Thr Lys Lys Glu Asn Arg Thr Pro Ser Tyr Ser Ser Val Ala Gln <210> 4 <211> 11 <212> PRT
<213> Bacillus licheniformis <400> 4 Ile Met Lys Thr Val Pro Ala Asp Lys Glu Ile <210> 5 <211> 11 <212> PRT
<213> Bacillus licheniformis <400> 5 His Tyr Val Ser Thr Leu Ser Glu Gln Glu Val <210> 6 <211> 15 <212> PRT
<213> Bacillus licheniformis <400> 6 Ala Tyr Gln Thr Ile Tyr His Leu Ser Glu Thr Val Gly Pro Arg <210> 7 <211> 22 <212> PRT
<213> Bacillus licheniformis <400> 7 Thr Ala Gly Gln Ile Ala Gly Thr Ala Val Tyr Asn Leu Thr Lys Lys Glu Asn Arg Thr Pro Ser <210> 8 <211> 27 <212> PRT
<213> Bacillus licheniformis <400> 8 Ala Tyr Gln Thr Ile Tyr His Leu Ser Glu Thr val Gly Pro Arg val Thr Gly Thr Ala Glu Glu Lys Lys Ser Ala Ala <210> 9 <211> 19 <212> PRT
<213> Bacillus licheniformis <400> 9 Lys Ala Lys Gly Val Lys Asn Pro Asp Ile Val Tyr Val Thr Ser His Tyr Asp Ser <210> 10 <211> 20 <212> PRT
<213> Bacillus licheniformis <400> 10 Gly Val Lys Asn Pro Asp Ile Val Tyr Val Thr Ser His Tyr Asp Ser Val Pro Tyr Ala <210> 11 <211> 20 <212> PRT
<213> Bacillus licheniformis <400> 11 Phe Ile Thr Phe Gly Ala Glu Glu Ile Gly Leu Leu Gly Ser Arg His Tyr Val Ser Thr <210> 12 <211> 20 <212> PRT
<213> Bacillus licheniformis <400> 12 Ala Ala Ser Leu Ser Leu Gly Lys Asp Val Leu Phe Leu His Gln Gly Gly Ser Ser Asp <210> 13 <211> 18 <212> DNA
<213> Artificial sequence <220>
<221> misc_feature <222> 6 <223> n can be any nucleotide <220>
<221> misc_feature <222> 15 <223> n can be any nucleotide <400> 13 aayccngaya thgtntay 18 <210> 14 <211> 18 <212> DNA
<213> Artificial sequence <220>
<221> misc_feature <222> 4 <223> n can be any nucleotide <220>
<221> misc_feature <222> 7 <223> n can be any nucleotide <220>
<221> misc_feature <222> 16 <223> n can be any nucleotide <400> 14 raanagnacr tcyttncc 18 <210> 15 <211> 6 <212> PRT
<213> Bacillus licheniformis <400> 15 Asn Pro Asp Ile Val Tyr <210> 16 <211> 7 <212> PRT
<213> Bacillus licheniformis <400> 16 Gly Lys Asp Val Leu Phe Leu <210> 17 <211> 5 <212> PRT
<213> Bacillus licheniformis <400> 17 Lys Phe Ser Lys Lys <210> 18 <211> 8 <212> PRT
<213> Bacillus licheniformis <400> 18 Asn val Glu Lys Phe Ser Lys Lys <210> 19 <211> 5 <212> PRT
<213> Bacillus licheniformis <400> 19 Asn Arg Thr Pro Ser

Claims (12)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An aminopeptidase having the amino acid sequence set forth in SEQ ID NO: 1.

2. An aminopeptidase comprising:

the amino acid sequence set forth in SEQ ID NO:1 having a deletion consisting of amino acid residues 1 through 30;
the amino acid sequence set forth in SEQ ID NO:1 having a deletion consisting of amino acid residues 1 through 39;
the amino acid sequence set forth in SEQ ID NO:1 having a deletion consisting of amino acid residues 1 through 40;

the amino acid sequence set forth in SEQ ID NO:1 having a deletion consisting of amino acid residues 1 through 41; or the amino acid sequence set forth in SEQ ID NO:1 having a deletion consisting of amino acid residues 1 through 42.

3. The aminopeptidase of claim 2, wherein the amino acid sequence set forth in SEQ ID NO:1 has a deletion consisting of amino acid residues 1 through 30.

4. The aminopeptidase of claim 2, wherein the amino acid sequence set forth in SEQ ID NO:1 has a deletion consisting of amino acid residues 1 through 39.

5. The aminopeptidase of claim 2, wherein the amino acid sequence set forth in SEQ ID NO:1 has a deletion consisting of amino acid residues 1 through 40.

6. The aminopeptidase of claim 2, wherein the amino acid sequence set forth in SEQ ID NO:1 has a deletion consisting of amino acid residues 1 through 41.

7. The aminopeptidase of claim 2, wherein the amino acid sequence set forth in SEQ ID NO:1 has a deletion consisting of amino acid residues 1 through 42.

8. An aminopeptidase having the amino acid sequence set forth in SEQ ID NO:1 having a deletion consisting of amino acid residues 444 through 449.

9. An isolated gene encoding the aminopeptidase according to any one of claims 1, 2, 3, 4, 5, 6 or 7.

10. An expression vector which contains a gene comprising the nucleotide sequence set forth in SEQ ID NO: 2, wherein said gene encodes an aminopeptidase.

11. An Escherichia coli transformant which is transformed with the expression vector deposited as accession number:
KCTC 1000 BP.

12. A process for preparing a naturally occurring protein, comprising the following steps: (1) purifying a recombinant protein containing a methionine-X-proline sequence at the amino terminus, wherein X of the methionine-X-proline sequence can be any kind of amino acid; (2) adding the aminopeptidase of any one of claims 1, 2, 3, 4, 5, 6 or 7 into the purified recombinant protein; and (3) digesting the methionine-X-proline sequence at the amino terminus of the recombinant protein by using the aminopeptidase.